New iridium-based complexes for ecl

ABSTRACT

Novel iridium-based Ir(III) luminescent complexes, conjugates comprising these complexes as a label and their application, for example in the electrochemiluminescence based detection of an analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/456,021 filed Aug. 11, 2014, which is a continuation of U.S.application Ser. No. 13/961,401 filed Aug. 7, 2013, which is acontinuation of International Application No. PCT/EP2012/051996 filedFeb. 7, 2012, which claims the benefit of European Patent ApplicationNo. 11153913.6 filed Feb. 9, 2011, the disclosures of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE DISCLOSURE

Electrogenerated chemiluminescence (also called electrochemiluminescenceand abbreviated ECL) is the process whereby species generated atelectrodes undergo high-energy electron-transfer reactions to formexcited states that emit light. The first detailed ECL studies weredescribed by Hercules and Bard et al. in the mid-1960s. After about 40years of study, ECL has now become a very powerful analytical techniqueand is widely used in the areas of, for example, immunoassay, food andwater testing, and biowarfare agent detection.

Various compounds appear to be of interest for use in organic lightemitting devices (OLEDs). These compounds may be appropriate for use insolid materials, for example, or may be dissolved in organic fluids.However, no conclusion can be drawn regarding their utility in anaqueous medium as, for example, required for detection of an analytefrom a biological sample.

In general ECL-based detection methods may be based on the use ofwater-soluble ruthenium complexes, comprising Ru(II+) as metal ion.Despite significant improvements made over the past decades, a needstill exists for more sensitive electrochemiluminescence-based in vitrodiagnostic assays.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to novel iridium-based Ir(II) luminescentcomplexes, conjugates comprising these complexes as a label and theirapplication, e.g. in the electrochemiluminescence based detection of ananalyte. As described herein, it has been surprisingly found thatcertain iridium-based If(III+) luminescent complexes, represent verypromising labels for suture high sensitive ECL-based detection methods.

According to some embodiments of the instant disclosure, aniridium-based chemiluminescent compound of Formula I is provided.Formula I:

wherein R1-R16 is hydrogen, halide, cyano- or nitro-group, amino,alkylamino, substituted alkylamino, arylamino, substituted arylamino,alkylammonium, substituted alkylammonium, carboxy, carboxylic acidester, carbamoyl, hydroxy, substituated or unsubstituated alkyloxy,substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or R17, wherein R17 is aryl,substituted aryl, alkyl, substituted alkyl branched alkyl, substitutedbranched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substitutedalkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,wherein the substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstituatedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or,wherein within R1-R12 or/and within R13-R16, respectively, two adjacentRs can form an aromatic ring or a substituted aromatic ring, wherein thesubstituent is selected from hydrogen, halide, cyano- or nitro-group, ahydrophilic group, like amino, alkylamino, substituted alkylamino,alkylammonium, substituted alkylammonium, carboxy, carboxylic acidester, carbamoyl, hydroxy, substituted or unsubstituted alkyloxy,substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or,wherein within R1-R12 or/and within R13-R16, respectively, two adjacentRs can form an aliphatic ring or a substituted aliphatic ring, whereinthe substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstitutedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate, andwherein at least one of R13-R16 is -Q-Y, wherein Q represents a linkerand Y is a functional group.

According to some embodiments, the present disclosure also discloses aconjugate comprising the above compound and covalently bound thereto anaffinity binding agent.

According to some embodiments, the present disclosure further relates tothe use of a compound or of a conjugate as disclosed herein forperforming a luminescence measurement or an electrochemiluminescencereaction in an aqueous solution, especially, in anelectro-chemiluminescent device or electrochemiluminescent detectionsystem.

According to some further embodiments, the present disclosure provides amethod for measuring an analyte by an in vitro method, the methodcomprising the steps of (a) providing a sample suspected or known tocomprise the analyte, (b) contacting said sample with a conjugateaccording to the present disclosure under conditions appropriate forformation of an analyte conjugate complex, and (c) measuring the complexformed in step (b) and thereby obtaining a measure of the analyte

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE DISCLOSURE

The embodiments disclosed herein are not intended to be exhaustive orlimit the disclosure to the precise form disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings.

The present disclosure relates to an iridium-based chemiluminescentcompound of Formula I

wherein R1-R16 is hydrogen, halide, cyano- or nitro-group, amino,alkylamino, substituted alkylamino, arylamino, substituted arylamino,alkylammonium, substituted alkylammonium, carboxy, carboxylic acidester, carbamoyl, hydroxy, substituated or unsubstituated alkyloxy,substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or R17, wherein R17 is aryl,substituted aryl, alkyl, substituted alkyl branched alkyl, substitutedbranched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substitutedalkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,wherein the substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstituatedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or, wherein within R1-R12 or/andwithin R13-R16, respectively, two adjacent Rs can form an aromatic ringor a substituted aromatic ring, wherein the substituent is selected fromhydrogen, halide, cyano- or nitro-group, a hydrophilic group, likeamino, alkylamino, substituted alkylamino, alkylammonium, substitutedalkylammonium, carboxy, carboxylic acid ester, carbamoyl, hydroxy,substituted or unsubstituted alkyloxy, substituted or unsubstitutedaryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfino, sulfeno,sulfonamide, sulfoxide, sulfodioxide, phosphonate, phosphinate or,wherein within R1-R12 or/and within R13-R16, respectively, two adjacentRs can form an aliphatic ring or a substituted aliphatic ring, whereinthe substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstitutedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate and wherein at least one ofR13-R16 is -Q-Y, wherein Q represents a linker and Y is a functionalgroup.

In some embodiments at least one of R1 to R16 of the compound accordingto Formula I is substituted by at least one hydrophilic group. Forexample, in some embodiments, illustrative substituents for substitutedalkyloxy include ethylenoxy chains comprising 1-40 ethylenoxy units, orcomprising 1-20 ethylenoxy units or comprising 1-10 ethylenoxy units.

Exemplary hydrophilic groups are amino, alkylamino, with alkyl meaning alinear chain such as methyl, ethyl, propyl, butyl pentyl chain or abranched alkyl chain such as isopropyl, isobutyl, tert. butyl, forexample a linear alkyl chain such as methyl or ethyl, substitutedalkylamino, this contains one or two for example a branched or linearchains bound to the N-atom, which are substituted by an additionalhydrophilic group such as hydroxyl or sulfo, in at least someembodiments this substituted alkylamino contains two hydroxypropyl orhydroxyethyl residues, arylamino, with aryl referring to an aromaticresidue, such as phenyl, or naphthyl, substituted arylamino, with arylas defined above and an additional residue formed by a hydrophilicgroup, alkylammonium, with alkyl as defined above and in someembodiments being a trimethylammonium residue or triethylammoniumresidue, subsituted alkylammonium, carboxy, carboxylic acid ester,including an alkyl ester such as methyl or ethyl ester, carbamoyl,hydroxy, substituated or unsubstituated alkyloxy with alkyl andsubstituted alkyl being as defined above or aryloxy or substitutedaryloxy wth aryl and substituted aryl being as defined above, sulfanyl,alkylsulfonyl, arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide,sulfoxide, sulfodioxide, phosphonate, phosphinate.

According to some embodiments, such hydrophilic group is selected fromamino, alkylamino, substituted alkylamino arylamino substitutedarylamino, alkylammonium, subsituted alkylammonium, carboxy, hydroxy,sulfo, sulfeno, sulfonamide, sulfoxide, sulfodioxide and phosphonate,where applicable, each preferably as defined in the above paragraph.

In some embodiments the hydrophilic group is selected from sulfo,sulfonamide, sulfodioxide. In an illustrative embodiment at least one ofthe groups R1 to R12 of Formula I is a sulfo group. In anotherillustrative embodiment at least one of R1 to R12 of thephenylphenantridine residues comprised in Formula I is substituted by atleast one hydrophilic group. In at least some embodiments, thephenylphenantridine residues comprised in Formula I are selected fromthe below given substituted phenylphenantridines.

In compounds according to the present disclosure the linker Q may have abackbone length of between 1 and 20 atoms. For example, the shortestconnection between the pyridyl ring of Formula I and the functionalgroup Y may consist of 1 to 20 atoms. In an exemplary embodiment, thelinker Q in the electrochemiluminescent complex of this disclosure is astraight or branched saturated, unsaturated, unsubstituted, substitutedC1-C20 alkyl chain, or a C1-C20 arylalkyl chain (wherein e.g. a phenylenring accounts for a length of four carbon atoms), or a 1 to 20 atomchain with a backbone consisting of carbon atoms and one or moreheteroatoms selected from O, N and S, or a 1 to 20 atom chain with abackbone consisting of carbon atoms and one or more heteroatoms selectedfrom O, N and S comprising at least one aryl, heteroaryl, substitutedaryl or substituted heteroaryl group (wherein e.g. a phenylen ringaccounts for a length of four atoms). In some embodiments the linker Qin a compound according to the present disclosure is a saturated C1-C12alkyl chain, or a C1-C12 arylalkyl chain, or a 1 to 12 atom chain with abackbone consisting of carbon atoms and one or more heteroatoms selectedfrom O, N and S, or a 1 to 12 atom chain with a backbone consisting ofcarbon atoms and one or more heteroatoms selected from O, N and Scomprising at least one aryl, heteroaryl, substituted aryl orsubstituted heteroaryl group (wherein e.g. a phenylen ring accounts fora length of four atoms).

In some embodiments the functional group Y comprised in theiridium-based complex according to the present disclosure is selectedfrom the group consisting of carboxylic acid, N-hydroxysuccinimideester, amino group, halogen, sulfhydryl, maleimido, alkynyl, azide, andphosphoramidite.

A conjugate comprising an iridium-based electrochemiluminescent compoundof Formula I, is disclosed and defined herein, and may be covalentlybound thereto a biological substance. Examples of suitable biologicalsubstances include cells, viruses, subcellular particles, proteins,lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids,peptidic nucleic acids (PNA), oligosaccharides, polysaccharides,lipopoly-saccharides, cellular metabolites, haptens, hormones,pharmacological substances, alkaloids, steroids, vitamins, amino acidsand sugars.

In some embodiments the biological substance of a conjugate according tothe present disclosure, i.e., covalently bound to a compound accordingFormula I is an affinity binding agent. As the skilled artisan willappreciate in a conjugate according to the present disclosure thefunctional group Y of the compound according to Formula I has been usedto form a covalent bond with a group on the affinity binding agent. Incase an affinity binding reagent would not in itself contain anappropriate group for binding or reacting with the group Y, such groupcan be easily introduced into the affinity binding agent by relying onwell-established procedures.

Not wishing to be limited further, but in the interest of clarity, theaffinity binding agent may comprise any of the following; an antigen, aprotein, an antibody, biotin or biotin analogue and avidin orstreptavidin, sugar and lectin, an enzyme, a polypeptide, an aminogroup, a nucleic acid or nucleic acid analogue and complementary nucleicacid, a nucleotide, a polynucleotide, a peptide nucleic acid (PNA), apolysaccharide, a metal-ion sequestering agent, receptor agonist,receptor antagonist, or any combination thereof. For example, theaffinity binding agent can be one partner of a specific binding pair,where the other partner of said binding pair is associated with or isthe target on a cell surface or an intracellular structure.

In some illustrative embodiments, an affinity binding agent may be apartner or member of an affinity binding pair, or as it is also calledby the skilled artisan, a partner or member of a specific binding pair.According to some embodiments, an affinity binding agent may hves atleast an affinity of 10⁷ I/mol to its target, e.g. one member of aspecific binding pair, like an antibody, to the other member of thespecific binding pair, like its antigen. An affinity binding agent mayalso have an affinity of 10⁸ I/mol or even more such as 10⁹ I/mol forits target.

In some exemplary embodiments the present disclosure relates to aconjugate wherein the affinity binding agent is selected from the groupconsisting of antigen, antibody, biotin or biotin analogue, avidin orstreptavidin, sugar, lectin, nucleic acid or nucleic acid analogue andcomplementary nucleic acid, receptor and ligand. In some furtherexemplary embodiments the present disclosure relates to a conjugatewherein the affinity binding agent is selected from the group consistingof antibody, biotin or biotin analogue, avidin or streptavidin, andnucleic acid. In further illustrative embodiments the conjugateaccording to the present disclosure comprises covalently linked acompound according to Formula I as disclosed and defined herein aboveand an affinity binding agent that either is an oligonucleotide or anantibody. Biotin analogues, according to the instant disclosure, includeaminobiotin, iminobiotin or desthiobiotin.

The term “oligonucleotide” or “nucleic acid” as used herein, generallyrefers to short, generally single stranded, polynucleotides thatcomprise at least 8 nucleotides and at most about 1000 nucleotides. Insome illustrative embodiments an oligonucleotide will have a length ofat least 9, 10, 11, 12, 15, 18, 21, 24, 27 or 30 nucleotides. In somefurther illustrative embodiments an oligonucleotide will have a lengthof no more than 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30nucleotides. The term oligonucleotide is to be understood broadly andincludes DNA and RNA as well as analogues and modification thereof.

A nucleic acid analogue may, for example, contain a substitutednucleotide carrying a substituent at the standard bases deoxyadenosine(dA), deoxyguanosine (dG), deoxycytosine (dC), deoxythymidine (dT),deoxyuracil (dU). Examples of such substituted nucleobases include:5-substituted pyrimidines like 5 methyl dC, aminoallyl dU or dC,5-(aminoethyl-3-acrylimido)-dU, 5-propinyl-dU or -dC, 5 halogenated-dUor -dC; N substituted pyrimidines like N4-ethyl-dC; N substitutedpurines like N6-ethyl-dA, N2-ethyl-dG; 8 substituted purines like8-[6-amino)-hex-1-yl]-8-amino-dG or -dA, 8 halogenated dA or dG, 8-alkyldG or dA; and 2 substituted dA like 2 amino dA. A nucleic acid analoguemay, for example, contain a nucleotide or a nucleoside analogue (e.g.,the naturally occurring nucleobases may be exchanged by using nucleobaseanalogs like 5-Nitroindol d riboside; 3 nitro pyrrole d riboside,deoxyinosine (dl), deoyxanthosine (dX); 7 deaza-dG, -dA, -dl or -dX;7-deaza-8-aza-dG, -dA, -dl or -dX; 8-aza-dA, -dG, -dl or -dX; dFormycin; pseudo dU; pseudo iso dC; 4 thio dT; 6 thio dG; 2 thio dT; isodG; 5-methyl-iso-dC; N8-linked 8-aza-7-deaza-dA; 5,6-dihydro-5-aza-dC;and etheno-dA or pyrollo-dC). As obvious to the skilled artisan, thenucleobase in the complementary strand should be selected in such amanner that duplex formation is specific. If, for example,5-methyl-iso-dC is used in one strand (e.g. (a)) iso dG should be in thecomplementary strand (e.g. (a′)). Further, in a nucleic acid analoguethe oligonucleotide backbone may be modified to contain substitutedsugar residues, sugar analogs, modifications in the internucleosidephosphate moiety, and/or be a PNA.

According to some embodiments, an oligonucleotide may for examplecontain a nucleotide with a substituted deoxy ribose like 2′-methoxy,2′-fluoro, 2′-methylseleno, 2′-allyloxy, 4′-methyl dN (wherein N is anucleobase, e.g., A, G, C, T or U).

Sugar analogs are, for example, Xylose; 2′,4′ bridged Ribose like (2′-O,4′-C methylene)-(oligomer known as LNA) or (2′-O, 4′-Cethylene)-(oligomer known as ENA); L-ribose, L-d-ribose, hexitol(oligomer known as HNA); cyclohexenyl (oligomer known as CeNA); altritol(oligomer known as ANA); a tricyclic ribose analog where C3′ and C5′atoms are connected by an ethylene bridge that is fused to acyclopropane ring (oligomer known as tricycloDNA); glycerin (oligomerknown as GNA); Glucopyranose (oligomer known as Homo DNA); carbaribose(with a cyclopentan instead of a tetrahydrofuran subunit);hydroxymethyl-morpholin (oligomers known as morpholino DNA)

A number of modifications of the internucleosidic phosphate moiety arealso known not to interfere with hybridization properties and suchbackbone modifications can also be combined with substituted nucleotidesor nucleotide analogs. Examples include phosphorthioate,phosphordithioate, phosphoramidate and methylphosphonateoligonucleotides.

PNA (having a backbone without phosphate and d-ribose) can also be usedas a DNA analog.

The above mentioned modified nucleotides, nucleotide analogs as well asoligonucleotide backbone modifications can be combined as desired in anoligonucleotide in the sense of the present disclosure.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (VH) followedby a number of constant domains. Each light chain has a variable domainat one end (VL) and a constant domain at its other end; the constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light-chain and heavy-chainvariable domains.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains generally contain the antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (K) and lambda (λ), based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequences of the constantdomains of their heavy chains, antibodies (immunoglobulins) can beassigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well knownand described generally in, for example, Abbas et al., Cellular and Mol.Immunology, 4th ed., W.B. Saunders, Co. (2000). An antibody may be partof a larger fusion molecule, formed by covalent or non-covalentassociation of the antibody with one or more other proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

“Antibody fragments” comprise a portion of an intact antibody, forexample, such as a portion comprising the antigen-binding regionthereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields a F(ab′)2 fragment that hastwo antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody-hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of an antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains that enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Plueckthun, In: The Pharmacology of Monoclonal Antibodies,Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994)pp. 269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP 0404097; WO 1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134;and Holliger, P. et al., PNAS USA 90 (1993) 6444-6448. Triabodies andtetrabodies are also described in Hudson, P. J. et al., Nat. Med. 9(2003) 129-134.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target-bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this disclosure. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal-antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal-antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

As mentioned, the compounds and conjugates as disclosed herein havequite favorable properties. For example the disclosed compounds orconjugates, respectively, show a high ECL efficiency. This highefficiency is also present if the corresponding measurements areperformed in an aqueous system as compared to many, many ECL-labels thatonly have shown high ECL-efficiency when analyzed in an organic solvent.E.g., many OLED dyes usually are analyzed in acetonitrile and either arenot soluble in an aqueous solution or, if soluble, due not showefficient electrochemiluminescence in an aqueous solution.

In some exemplary embodiments the present disclosure relates the use ofa compound or of a conjugate, respectively, as disclosed in the presentdisclosure for performing an electrochemiliuminescense reaction in anaqueous solution. According to such embodiments, an aqueous solution isany solution comprising at least 90% water (weight by weight). Obviouslysuch aqueous solution may contain in addition ingredients like buffercompounds, detergents and for example tertiary amines liketripropylamine as electron donor in the ECL reaction.

In some embodiments, the present disclosure relates to the use of acompound or of a conjugate, respectively, as disclosed in the presentdisclosure in an electrochemiluminescence based detection method. Insome embodiments the present disclosure relates the use of a compound orof a conjugate, respectively, as disclosed in the present disclosure inthe detection of an analyte.

An analyte according to the present disclosure may be any inorganic ororganic molecule, including any biological substance of interest.Examples of suitable biological substances that represent an analyte inthe sense of the present disclosure are cells, viruses, subcellularparticles, proteins, lipoproteins, glycoproteins, peptides,polypeptides, nucleic acids, oligosaccharides, polysaccharides,lipopoly-saccharides, cellular metabolites, haptens, hormones,pharmacological substances, alkaloids, steroids, vitamins, amino acidsand sugars. According to the instant disclosure, the analyte may beselected from the group consisting of a polypeptide, a carbohydrate, andan inorganic or organic drug molecule.

A polypeptide or protein is a molecule that is essentially composed ofamino acids and that has at least two amino acids linked by peptidiclinkage. In case the analyte of interest to be investigated in a methoddisclosed here, the polypeptide may consist of at least 5, 6, 7, 8, 9,10, 12, 15, 20, 25, and 30 to up to about 10,000 amino acids. Forexample, according to some embodiments the polypeptide contains from 5to 2,000, or from 10 to 1,000 amino acids.

In case the analyte is a nucleic acid, these nucleic acids may comprisenaturally occurring DNA or RNA oligonucleotides.

In some embodiments the present disclosure relates to a method formeasuring an analyte by an in vitro method, the method comprising thesteps of (a) providing a sample suspected or known to comprise theanalyte, (b) contacting said sample with a conjugate according betweenan affinity binding agent and a compound according to Formula I asdisclosed in the present disclosure under conditions appropriate forformation of an analyte conjugate complex, (c) measuring the complexformed in step (b) and thereby obtaining a measure of the analyte.

In some embodiments the measurement in the above method for detection ofan analyte is performed by using an electrochemiluminescence baseddetection procedure. Also, in at least some such embodiments, the methodis practiced in an aqueous solution.

The following examples, sequence listing, and FIGURES are provided forthe purpose of demonstrating various embodiments of the instantdisclosure and aiding in an understanding of the present disclosure, thetrue scope of which is set forth in the appended claims. These examplesare not intended to, and should not be understood as, limiting the scopeor spirit of the instant disclosure in any way. It should also beunderstood that modifications can be made in the procedures set forthwithout departing from the spirit of the disclosure.

ILLUSTRATIVE EMBODIMENTS

The following comprises a list of illustrative embodiments according tothe instant disclosure which represent various embodiments of theinstant disclosure. These illustrative embodiments are not intended tobe exhaustive or limit the disclosure to the precise forms disclosed,but rather, these illustrative embodiments are provided to aide infurther describing the instant disclosure so that others skilled in theart may utilize their teachings.

1. An iridium-based chemiluminescent compound of Formula I

wherein R1-R16 is hydrogen, halide, cyano- or nitro-group, amino,alkylamino, substituted alkylamino, arylamino, substituted arylamino,alkylammonium, substituted alkylammonium, carboxy, carboxylic acidester, carbamoyl, hydroxy, substituated or unsubstituated alkyloxy,substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or R17, wherein R17 is aryl,substituted aryl, alkyl, substituted alkyl branched alkyl, substitutedbranched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substitutedalkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,wherein the substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstituatedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or,wherein within R1-R12 or/and within R13-R16, respectively, two adjacentRs can form an aromatic ring or a substituted aromatic ring, wherein thesubstituent is selected from hydrogen, halide, cyano- or nitro-group, ahydrophilic group, like amino, alkylamino, substituted alkylamino,alkylammonium, substituted alkylammonium, carboxy, carboxylic acidester, carbamoyl, hydroxy, substituted or unsubstituted alkyloxy,substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or,wherein within R1-R12 or/and within R13-R16, respectively, two adjacentRs can form an aliphatic ring or a substituted aliphatic ring, whereinthe substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstitutedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate andwherein at least one of R13-R16 is -Q-Y, wherein Q represents a linkerand Y is a functional group.2. The compound according to embodiment 1, wherein the linker Q is astraight or branched saturated, unsaturated, unsubstituted orsubstituted C1-C20 alkyl chain, or a 1 to 20 atom chain with a backboneconsisting of carbon atoms and one or more heteroatoms selected from O,N and S.3. The compound according to embodiment 1, wherein the linker Q is asaturated C1-C12 alkyl chain or a 1 to 12 atom chain with a backboneconsisting of carbon atoms and one or more heteroatoms selected from O,N and S.4. The compound according to embodiment 1 or 2, wherein the functionalgroup Y is selected from the group consisting of carboxylic acid,N-hydroxysuccinimide ester, amino group, halogen, sulfhydryl, maleimido,alkynyl, azide and phosphoramidite.5. A conjugate comprising a compound according to any of embodiments 1to 4 and covalently bound thereto an affinity binding agent.6. The conjugate of embodiment 5, wherein the affinity binding agent isselected from the group consisting of antigen and antibody, biotin orbiotin analogue and avidin or streptavidin, sugar and lectin, nucleicacid or nucleic acid analogue and complementary nucleic acid andreceptor and ligand.7. The conjugate according to embodiment 5 or 6, wherein said affinitybinding agent is a nucleic acid or an antibody.8 Use of a compound according to any of embodiments 1 to 4 or of aconjugate according to any of embodiments 5 to 7 for performing anelectrochemiluminescence reaction in an aqueous solution.9. Use of a compound according to any of embodiments 1 to 4 or of aconjugate according to any of embodiments 5 to 7 in anelectrochemiluminescence based detection method.10. Use of a compound according to any of embodiments 1 to 4 or of aconjugate according to any of embodiments 5 to 7 in the detection of ananalyte.11. A method for measuring an analyte by an in vitro method, the methodcomprising the steps ofa) providing a sample suspected or known to comprise the analyteb) contacting said sample with a conjugate according to any ofembodiments 5 to 7 under conditions appropriate for formation of ananalyte conjugate complex,c) measuring the complex formed in step (b) and thereby obtaining ameasure of the analyte.

EXAMPLES Example 1 Synthesis of Substituted Phenyl-PhenanthridinesExample 1.1 General Procedure for the Synthesis of Substituted2-Aminobiphenyls

With the Suzuki-Miyaura coupling reaction as described by Youn, S. W.,in Tetrahedron Lett. 50 (2009) 4598-4601 between commercially available2-bromoaniline derivates and the corresponding arylboronic acid theappropriate 2-aminobiphenyls can be synthesized, which are required forfurther reactions to phenanthridines.

Typical Procedure:

a: 10 mol % PdCl₂(PPh₃)₂, K₂CO₃, DMF/H₂O (5/1), 80° C., 24 h

Other Examples

Example 1.2 General Procedure for the Synthesis of SubstitutedPhenanthridines

To the ice-cooled solution of 2-arylaniline 1 (0.01 mol) in chloroform(20 ml) was added aryl acid chloride 2 (0.01 mol) and stirred underinert condition for 30 min at room temperature. The resulting mixturewas refluxed with stirring for the next 2 hours. The reaction mixturewas treated by the dropwise addition of pyridine (0.02 mol in 10 mlchloroform) over a period of 60 minutes. The mixture was allowed to coolto room temperature and stirred overnight. The mixture was washed wellwith 0.5 M HCl, dried over MgSO₄ and concentrated in vacuum. The crudeproduct was purified by flash chromatography on silica gel, 3:2hexane/ethyl acetate to give pure product 3 in 66% yield.

Benzamido-2-biphenyl 3 (0.01 mol) and POCl₃ (5 ml) in 20 ml of toluenewere refluxed and stirred under nitrogen for 18 hours, following theprocedure described by Lion, C., in Bull. Soc. Chim. Belg. 98 (1989)557-566. The cooled reaction mixture was diluted with CH₂Cl₂ (30 ml) andpoured into ice, washed with 25% NH₄OH and distilled water. The organiclayer was dried over MgSO₄ and concentrated in vacuo, followed by flashchromatography (silica gel, 1:1 hexane/ethyl acetate) gave the product4,6-phenylphenanthridine.

Yield: 52%. White solid. ¹H NMR (CDCl₃, 400 MHz) δ 7.54-7.85 (m, 9H),8.10 (d, J=8.0 Hz, 1H), 8.28 (d, J=7.9 Hz, 1H), 8.62 (d, J=8.4 Hz, 1H),8.67 (d, J=8.4 Hz, 1H).

Using 2-naphthalen-2-yl-phenylamine instead of 2-aryl-aniline

¹H-NMR (400 MHz, CDCl₃) δ 8.64 (d, J=9.1 Hz, 2H), 8.29 (d, J=8.1 Hz,1H), 8.16 (d, J=8.92 Hz, 1H), 7.92 (d, J=7.48 Hz, 1H), 7.79-7.75 (m,2H), 7.69 (t, J=14.0, 8.2 Hz, 1H), 7.63-7.61 (m, 2H), 7.53-7.46 (m, 4H),7.19 (t, J=14.3, 7.2 Hz, 1H).

MS: [M+H]⁺ 306.3

Using naphthalene-carbonyl chloride instead of phenyl acid chloride:

¹H-NMR (400 MHz, CDCl₃) δ 8.74 (d, J=8.3 Hz, 1H), 8.65 (d, J=8.1 Hz,1H), 8.27 (d, J=8.1 Hz, 1H), 8.23 (s, 1H), 8.15 (d, J=8.3 Hz, 1H), 8.03(d, J=8.4 Hz, 1H), 7.97-7.94 (m, 2H), 7.90-7.85 (m, 2H), 7.80-7.69 (m,2H), 7.62 (t, J=14.2, 7.1 Hz, 1H), 7.59-7.55 (m, 2H)

MS: [M+H]⁺306.3

Example 1.3 Procedure for the Synthesis of 6-(2-Sulfophenyl)Phenanthridine

The 6-(2-sulfophenyl)phenanthridine can be synthesized by gentle heatingof arylaniline (0.01 mol) with 2-sulfobenzoic acid cyclic anhydride(0.01 mol) in CH₃CN for 6 hours using the procedure as described byNicolai, E., in Chem. Pharm. Bull. 42 (1994) 1617-1630.

After purification the product can be converted to the appropriatephenanthridine based on the method described in example 1.2.

Example 1.4 Procedure for the Synthesis of 6-Phenyl-AlkylsulfonylPhenanthridine

The 6-phenyl-alkylsulfonyl phenanthridine can be synthesized by gentleheating of alkylsulfonyl-arylaniline (0.01 mol) with benzoic acidchloride (0.01 mol) in chloroform using the procedure as described byLion, C., in Bull. Soc. Chim. Belg. 98 (1989) 557-566, see example 1.2.

After purification the product can be converted to the appropriatephenanthridine based on the method described in example 1.2.

1H-NMR (400 MHz, CDCl3) δ 8.92 (d, J=8.7 Hz, 1H), 8.75 (d, J=1.9 Hz,1H), 8.68 (d, J=7.0 Hz, 1H), 8.35 (dd, J=8.7, 2.0 Hz, 1H), 8.30 (d,J=7.2 Hz, 1H), 7.89 (t, J=15.3, 7.1 Hz, 1H), 7.81-7.73 (m, 3H),7.64-7.56 (m, 3H) 3.12 (s, 3H).

MS: [M+H]+ 334.3

The 6-(4-methylsulfophenyl)phenanthridine can be also prepared byfollowing the procedure described by Cymerman, J., in J. Chem. Soc.(1949) 703-707.

Example 1.5 Synthesis of6-[4-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-phenyl]-phenanthridine

Synthesis of 2,5,8,11-tetraoxatridecan-13-ol tosylate

Procedure: (JACS, 2007, 129, 13364) To a solution of2,5,8,11-tetraoxatridecan-13-ol (7 g, 33.6 mmol) and triethylamine (4.9ml, 35.3 mmol) in dry CH2Cl2 (100 ml), 4-toluenesulfonyl chloride (6.7g, 35.3 mmol) and DMAP (120 mg) were added. The mixture was stirred atroom temperature for 20 h. The reaction mixture was washed with 80 mL ofHCl (1 M) and then water. The extract was dried over anhydrous MgSO4,filtrated, and the filtrate was evaporated. The residue was used in thenext step without further purification. Yield: 11.0 g (90%)

NMR: ¹H NMR (400 MHz, CDCl₃) δ 7.75-7.64 (m, 2H), 7.31-7.26 (m, 2H),4.16-4.06 (m, 2H), 3.62 (m 2H), 3.59-3.40 (m, 10H), 3.30 (s, 3H), 2.38(s, 3H).

¹³C{¹H}NMR (101 MHz, CDCl₃) δ 144.75 (s), 132.90 (s), 129.77 (s), 127.8(s), 71.82 (s), 70.60 (s), 70.48 (s), 70.47 (s), 70.41 (s), 70.39 (s),69.23 (s), 68.55 (s), 58.90 (s), 21.53 (s).

Synthesis of 4-PEG4-benzoic acid ethyl ester

Procedure: (JACS, 2007, 129, 13364) A mixture of compound ethyl2,5,8,11-tetraoxatridecan-13-yl 4-methylbenzenesulfonate (8.1 g, 22.3mmol), 4-hydroxtybenzoic acid ethyl ester (3.7 g, 22.3 mmol), K2CO3(15.4 g, 111.5 mmol) and 18-crown-6 (0.59 g, 2.2 mmol) was refluxed inacetone (120 ml) for 22 h. The reaction mixture was concentrated andextracted with ethyl acetate. The extract was washed with H2O, driedover anhydrous MgSO4, and filtrated. The filtrate was evaporated todryness and the residue was purified by column chromatography on silicagel (dichloromethane/methanol=100:1) to obtain the compound (1.93 g,88%). Yield: 7 g (88%)

NMR: ¹H NMR (400 MHz, CDCl₃) δ 8.01-7.84 (m, 2H), 6.96-6.85 (m, 2H),4.29 (q, J=7.1 Hz, 2H), 4.12 (dd, J=5.4, 4.3 Hz, 2H), 3.82 (dd, J=5.4,4.2 Hz, 2H), 3.71-3.56 (m, 10H), 3.51-3.45 (m, 2H), 3.32 (s, 3H), 1.32(t, J=7.1 Hz, 3H).

¹³C{¹H}NMR (101 MHz, CDCl3) δ 166.29 (s), 162.47 (s), 131.45 (s), 123.01(s), 114.11 (s), 71.90 (s), 70.84 (s), 70.60 (s), 70.59 (s), 70.58 (s),70.48 (s), 69.51 (s), 67.54 (s), 60.57 (s), 58.98 (s), 14.35 (s).

NMS(+): [M+Na⁺]⁺=calc. 379.1727. found 379.1743.

Synthesis of 4-PEG4-benzoic acid

Procedure: (JACS, 2007, 129, 13364) A mixture of compound ethyl4-(2,5,8,11-tetraoxatridecan-13-yloxy)benzoate (7 g, 19.6 mmol), and KOH(2.3 g, 41.24 mmol) in 200 mL of EtOH/H2O (1:1 v/v) was refluxovernight. After cooling down, the mixture was neutralized with HCl(2N). The resulting mixture was extracted with EtOAc and evaporated todryness. The resulting white solid was recrystallized in EtOAc/hexanes.Yield: 5.3 g (85%)

NMR: ¹H NMR (300 MHz, CDCl₃) δ 11.17 (s, 1H), 8.14-7.89 (m, 2H),7.03-6.75 (m, 2H), 4.29-4.02 (m, 2H), 3.92-3.81 (m, 2H), 3.78-3.57 (m,10H), 3.57-3.46 (m, 2H), 3.35 (s, 3H).

¹³C{¹} NMR (75 MHz, CDCl3) δ 171.46 (s), 163.24 (s), 132.30 (s), 121.98(s), 114.33 (s), 71.96 (s), 70.91 (s), 70.67 (s), 70.66 (s), 70.64 (s),70.54 (s), 69.55 (s), 67.66 (s), 59.08 (s)

MS(−): [M−H]⁻=calc. 327.1438. found 327.1456.

Synthesis ofN-Biphenyl-2-yl-4-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzamide

Procedure: To a solution of4-(2,5,8,11-tetraoxatridecan-13-yloxy)benzoic acid (3 g, 9.14 mmol), 0.2mL of DMF in 30 mL dry DCM at 0° C., oxalyl chloride (1.05 mL, 12.34mmol) was added. The reaction mixture was stirred at 0° C. for 1 h. Thesolution was concentrated to dryness. The oily residue was used withoutfurther purification in the next step.

A solution of 2-phenylaniline (1.6 g), pyridine (2.4 mL) in chloroform(80 mL) under inert atmosphere was cooled down to 0° C.(phenyl-4-(2,5,8,11-tetraoxatridecan-13-yloxy)benzoyl chloride (3.1 g,9.14 mmol) in 20 mL was slowly added to the solution and the finalmixture allowed to reach room temperature. The solution was reflux for 2h and stirred overnight at room temperature. The reaction mixture wasextracted with HCl (1 M, 2×100 mL), NaHCO3 (100 mL) and water (50 mL).The organic phase was dried with MgSO4 and purified by chromatography insilica gel (EtOAc/hexane). Yield: 4.1 (90%)

NMR: ¹H NMR (400 MHz, CDCl₃) δ 8.49 (dd, J=8.3, 0.9 Hz, 1H), 7.94 (s,1H), 7.61-7.35 (m, 9H), 7.33-7.25 (m, 1H), 7.19 (m, 1H), 6.91-6.84 (m,2H), 4.16-4.10 (m, 2H), 3.85 (m, 2H), 3.77-3.58 (m, 10H), 3.56-3.49 (m,2H), 3.36 (s, 3H)

¹³C{¹H}NMR (101 MHz, CDCl3) δ 164.56 (s), 161.65 (s), 138.18 (s), 135.12(s), 132.32 (s), 129.97 (s), 129.39 (s), 129.22 (s), 128.66 (s), 128.57(s), 128.16 (s), 127.13 (s), 124.18 (s), 121.23 (s), 114.57 (s), 71.95(s), 70.89 (s), 70.64 (s), 70.63 (s), 70.54 (s), 69.54 (s), 67.63 (s),59.04 (s), 53.51 (s).

MS(+): [M+H]⁺=calc. 480.2386 found. 480.2383; [M+Na]⁺=calc. 502.2200.found 502.2204.

Synthesis of6-[4-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-phenyl]-phenanthridine

Procedure:N-Biphenyl-2-yl-4-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzamide(4 g, 8.34 mmol), POCl3 (10 ml) in 10 ml toluene were refluxed for 20 h.The mixture was cooled down to room temperature, and 100 ml ofdichloromethane were added. The solution was poured into ice and themixture neutralized with NH4OH (20%). The organic phase was extractedand washed successively with destilled water and brine, and dried overMgSO4. The resulting solution was purified by flash chromatography(silica gel, in ethyl acetate/hexane 1:1, Rf=0.14). Yield: 1 g (25%)

NMR: ¹H NMR (300 MHz, CDCl₃) δ 8.68 (d, J=8.3 Hz, 1H), 8.59 (dd, J=8.1,1.4 Hz, 1H), 8.23 (dd, J=8.1, 1.1 Hz, 1H), 8.15 (dd, J=8.3, 0.7 Hz, 1H),7.84 (ddd, J=8.3, 7.1, 1.3 Hz, 1H), 7.79-7.57 (m, 5H), 7.15-7.03 (m,2H), 4.29-4.19 (m, 2H), 3.93-3.90 (m, 2H), 3.80-3.60 (m, 12H), 3.59-3.49(m, 2H), 3.37 (s, 3H).

¹³C{¹H}NMR (75 MHz, CDCl3) δ 160.92 (s), 159.45 (s), 143.84 (s), 133.59(s), 131.26 (s), 130.61 (s), 130.26 (s), 129.05 (s), 128.90 (s), 127.19(s), 126.85 (s), 125.39 (s), 123.70 (s), 122.29 (s), 122.01 (s), 114.68(s), 72.02 (s), 70.97 (s), 70.74 (s), 70.72 (s), 70.69, 70.62 (s), 69.80(s), 67.68 (s), 59.15 (s).

MS (+) JM358-F5, [M+H]⁺ calc=462.2280. found 462.2275.

Example 2 General Procedure for the Synthesis of Chloro-Cross-LinkedDimer Complex

The general procedure was published by Nonoyama, M., J. Organomet. Chem.86 (1975) 263-267.

The iridium dimers were synthesized as follow: IrCl₃.3H₂O and 2.5 equivof 6-phenylphenanthridine were heated at 120° C. for 18 h under nitrogenin 2-ethoxyethanol/water mixture (3:1, v/v). After being cooled to roomtemperature the precipitate was filtered off and successively washedwith methanol and Et₂O, dried to afford the desired dimer.

Example 2.1 Complex with Unsubstituted Phenylphenanthridine

[(6-phenylphenanthridine)2IrCl]2

Yield: 71%. Brown solid. ¹H NMR (DMSO-d₆, 400 MHz) δ 6.45 (d, J=6.8,4H), 6.58 (t, J=7.1, 13.9 Hz, 4H), 6.95 (t, J=7.1, 14.2 Hz, 4H), 7.56(t, J=7.4, 16.0 Hz, 4H), 7.68 (t, J=8.1, 16.2 Hz, 4H), 7.93 (t, J=8.0,14.6 Hz, 4H), 8.07-8.13 (m, 8H), 8.80 (d, J=7.3 Hz, 4H), 8.93-9.01 (m,12H).

Example 2.2 Complex with Substituted Phenylphenanthridine

A mixture of6-[4-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-phenyl]-phenanthridine(1 g, 2.16 mmol), IrCl₃.3H₂O (346 mg, 0.98 mmol) in 16 ml of2-EtOEtOH:H₂O (12:4) was refluxed overnight under nitrogen atmosphere.The reaction mixture was cooled down to room temperature and 60 ml ofwater were added to obtain an oily precipitate. The supernadant wasdiscarded and 50 ml of water were added to the residue. The mixture wasstirred for 1 h to obtain a red-brownish precipitate. The solid wasfiltrated and washed with water (50 ml) and Et₂O (30 ml). The brownsolid was dissolved in the smaller amount of dichloromethane andprecipitated upon addition of Et₂O. It was used in the next step withoutfurther purification. Yield: 550 mg (50%)

NMR: ¹H NMR (300 MHz, CDCl3) δ 8.74 (d, J=8.1 Hz, 4H), 8.36 (dd, J=8.0,5.2 Hz, 8H), 7.90 (dd, J=14.7, 7.7 Hz, 8H), 7.81 (d, J=9.0 Hz, 4H),7.79-7.67 (m, 4H), 6.78-6.65 (m, 4H), 6.32 (dd, J=8.8, 2.5 Hz, 4H),5.89-5.83 (m, 4H), 5.28 (d, J=2.5 Hz, 4H), 3.67-3.10 (m, 100H, PEGChain, contains some impurities)

MS(ESI-MS(+)): [M+2Na⁺]²⁺ calc. 1171.3463. found 1171.3473.[(ĈN)₂Ir]=calc. 1113.3877. found 1113.3892.

Example 3 A) Synthesis of Carboxyalkylenoxy-Picolinic Acid Derivatives

A mixture of the 3-hydroxy-2-pyridinecarboxylic acid (0.01 mol), theethyl 4-bromobutanoate or ethyl 6-bromohexanoate (0.021 mol), and amixture of potassium carbonate (5 eq.) in DMF (20 ml) was heated at 90°C. for 20 hours under nitrogen. After cooling, the reaction mixture waspoured into ice-water mixture and extracted three times withdichloromethane (30 ml), dried over anhydrous MgSO₄, filtered, and thesolvent was evaporated to dryness. Purified by flash chromatography(silica, hexane/ethyl acetate 3:1) to afford the product (based on U.S.Pat. No. 5,219,847).

The formed ester was hydrolyzed by NaOH in MeOH (pH=10). The pH of thesolution was then adjusted to 6.0 and stirred at r.t. overnight. Thesolvent was removed in vacuo and the residue was crystallized fromhexane/acetone to give the desired product.

3-(Carboxy-pentyloxy)-pyridine-2-carboxylic acid. Yield: 51%. Graysolid. ¹H NMR (DMSO-d₆, 400 MHz) δ1.39-1.45 (m, 2H), 1.51-1.57 (m, 2H),1.67-1.74 (m, 2H), 2.19-2.23 (m, 2H), 4.04-4.07 (m, 2H), 7.47-7.50 (m,1H), 7.61 (d, J=8.1 Hz, 1H), 8.13 (d, J=8.1 Hz, 1H).

B) Synthesis of 5-[4-(2-Carboxy-ethyl)-phenyl]-pyridine-2-carboxylicacid

Under an argon atmosphere, to 4 ml 1,2-dimethoxyethane are added5-bromo-pyridine-2-carboxylic acid (93 mg, 0.46 mmol),4-(2-carboxyethyl)benzeneboronic acid (106 mg, 0.55 mmol), 0.51 ml of a2M aqueous sodium carbonate solution and dichlorobis-(triphenylphosphin)palladium (II) (20 mg, 0.03 mmol). The mixture is stirred at 90° C.overnight, cooled and quenched with water. Ethyl acetate is added andthe mixture adjusted to pH=2 with 1 M hydrochloric acid. After threefoldextraction with ethyl acetate, the combined organic layers are driedover magnesium sulfate, filtered, and evaporated in vacuo. The residueis purified by silica gel chromatography (eluent:dichloromethane/methanol 5:1).

¹H-NMR (400 MHz, DMSO-d₆) δ 9.00 (d, J=2 Hz, 1H), 8.25 (dd, J=8.2, 2.3Hz, 1H), 8.11 (d, J=8.1 Hz, 1H), 7.74 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.2Hz, 2H), 2.91 (t, J=15, 7.5 Hz, 2H), 2.61 (t, J=15.1, 7.6 Hz, 2H)

MS: [M+H]⁺ 272.3

Example 4 General Procedure for the Synthesis of Iridium Complexes

A chloro-cross-linked dimer complex 0.5 mmol, picolinate 1.25 mmol andNa₂CO₃ 3 mmol were mixed into 2-ethoxyethanol (12 ml) and heated at 120°C. for 15 hours. To the cooled mixture distilled water was added (25ml), the crude product was then filtered off and washed with water,followed by portions of n-hexane and Et₂O. The product was purified bycolumn chromatography (silica, n-hexane/dichloromethane) to give a redpowder. (based on Lamansky, S., Inorg. Chem. 40 (2001) 1704-1711)

Ir(6-phenylphenanthridine)₂Pyridine-2-carboxylic acid

¹H NMR (400 MHz, CDCl₃) δ 9.17 (d, J=7.8 Hz, 1H), 9.09 (d, J=8.2 Hz,1H), 8.71 (d, J=8.2 Hz, 1H), 8.62 (t, J=14.8, 7.8 Hz, 2H), 8.43-8.33 (m,4H), 8.23 (d, J=8.1 Hz, 1H), 7.92-7.77 (m, 4H), 7.65 (t, J=15, 7.9 Hz,2H), 7.57-7.46 (m, 3H), 7.36 (t, J=14.8, 7.8 Hz, 1H), 7.19-7.16 (m, 2H),7.10 (d, J=7.8 Hz, 1H), 7.04 (t, J=14.2, 6.8 Hz, 1H), 6.92 (t, J=14.1,6.7 Hz, 1H), 6.80 (t, J=13.7, 6.8 Hz, 1H), 6.67 (t, J=13.7, 6.6 Hz, 1H),6.51 (d, J=6.8 Hz, 1H).

MS: [M+H]⁺ 826.4

Ir(6-phenylphenanthridine)₂ 5-(Methoxy)pyridine-2-carboxylic acid

¹H-NMR (400 MHz, CDCl3) δ 9.15 (d, J=8.2 Hz, 1H), 9.09 (d, J=8.2 Hz,1H), 8.70 (d, J=7.8 Hz, 1H), 8.61 (d, J=8.2 Hz, 2H), 8.44-8.35 (m, 3H),8.21 (d, J=8.0 Hz, 1H), 7.97 (d, J=2.7, 1 H), 7.91-7.86 (m, 2H),7.82-7.80 (m, 2H), 7.68 (d, J=8.6 Hz, 1H), 7.57-7.53 (m, 3H), 7.36 (t,J=15.2, 7.2 Hz, 1H), 7.14 (t, J=15.1, 7.6 Hz, 1H), 7.08-6.93 (m, 4H),6.78 (t, J=14.9, 7.6 Hz, 1H), 6.65 (t, J=14.8, 7.6 Hz, 1H), 6.49 (d,J=7.6 Hz, 1H), 3.63 (s, 3H)

MS: [M+H]⁺ 854.2

Ir(6-phenylphenanthridine)₂ 4-(Hydroxymethyl)pyridine-2-carboxylic acid

¹H-NMR (400 MHz, DMSO-d₆) δ 9.14 (d, J=8.1 Hz, 2H), 8.96 (d, J=8.0 Hz,1H), 8.87 (d, J=7.7 Hz, 1H), 8.73 (d, J=7.7 Hz, 1H), 8.68 (d, J=7.8 Hz,1H), 8.51 (d, J=8.6 Hz, 1H), 8.37 (d, J=8.2 Hz, 1H), 8.26-8.24 (m, 2H),8.10 (t, J=14.7, 7.3 Hz, 1H), 8.02-7.96 (m, 3H), 7.68 (d, J=8.4 Hz, 1H),7.62 (t, J=15.2, 7.1 Hz, 1H), 7.53-7.48 (m, 2H), 7.39-7.37 (m, 2H), 7.16(t, J=15.3, 7.2 Hz, 1H), 7.10-7.04 (m, 2H), 6.86 (d, J=6.8 Hz, 1H), 6.78(t, J=14.2, 7.1 Hz, 1H), 6.67 (t, J=14.9, 7.3 Hz, 1H), 6.35 (d, J=6.8Hz, 1H), 5.32 (s, 1H), 4.33 (s, 2H).

MS: [M+H]⁺ 854.2

Ir(6-phenylphenanthridine)₂ 3-Hydroxypyridine-2-carboxylic acid:′

¹H-NMR (400 MHz, CDCl₃) δ 9.15 (d, J=8.3 Hz, 1H), 9.06 (d, J=8.2 Hz,1H), 8.65-8.57 (m, 3H), 8.46-8.41 (m, 2H), 8.34 (d, J=8.0 Hz, 1H), 8.21(d, J=8.0 Hz, 1H), 7.94-7.78 (m, 5H), 7.72 (d, J=7.8 Hz, 1H), 7.58-7.55(m, 2H), 7.40 (t, J=14.0, 7.0 Hz, 1H), 7.15 (t, J=15.2, 7.0 Hz, 1H),7.05-6.95 (m, 5H), 6.77 (t, J=13.7, 7.0 Hz, 1H), 6.66 (t, J=13.6, 6.4Hz, 1H), 6.50 (d, J=6.6 Hz, 1H).

MS: [M+H]⁺ 839.2

Ir(6-phenyl-benzophenanthridine)₂Pyridine-2-carboxylic acid

¹H NMR (400 MHz, CDCl₃) δ 9.04 (m, 4H), 8.82 (m, 2H), 8.77-8.70 (m, 1H),8.41 (d, J=8.0 Hz, 1H), 8.29-8.27 (m, 2H), 8.15-8.09 (m, 4H), 7.85 (d,J=8.3 Hz, 1H), 7.78-7.71 (m, 4H), 7.65 (d, J=7.7 Hz, 1H), 7.62-7.553 (m,2H), 7.45-7.40 (m, 2H), 7.23-7.17 (m, 1H), 7.13-7.05 (m, 3H), 7.05-7.00(m, 1H), 6.83 (dd, J=10.8, 4.0 Hz, 1H), 6.68 (dd, J=10.9, 3.8 Hz, 1H),6.51 (dd, J=7.6, 0.9 Hz, 1H).

MS: [M+H]⁺ 924.2

Ir(6-phenylphenanthridine)₂ 2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid

¹H-NMR (400 MHz, DMSO-d₆) δ 9.24 (m, 1H), 9.15 (d, J=8.0 Hz, 1H), 8.97(d, J=8.4 Hz, 1H), 8.88 (d, J=8.1 Hz, 1H), 8.73 (d, J=7.6 Hz, 1H), 8.68(d, J=7.4 Hz, 1H), 8.50 (d, J=7.8 Hz, 1H), 8.45 (d, J=7.9 Hz, 1H), 8.34(d, J=2.0 Hz, 1H), 8.28 (d, J=7.9 Hz, 1H), 8.13-8.00 (m, 4H), 7.92 (dd,J=8.1, 2.1 Hz, 1H), 7.63 (t, J=15.2, 7.0 Hz, 2H), 7.54-7.42 (m, 3H),7.35 (d, J=8.2 Hz, 2H), 7.17 (t, J=15.2, 7.0 Hz, 1H), 7.10-7.06 (m, 3H),7.02 (t, J=15.7, 7.3 Hz, 1H), 6.89 (d, J=6.7 Hz, 1H), 6.77 (t, J=14.0,7.1 Hz, 1H), 6.71 (t, J=14.8, 7.0 Hz, 1H), 6.45 (d, J=6.7 Hz, 1H), 2.86(t, J=15.2, 7.5 Hz, 2H), 2.55 (t, J=15.4, 7.7 Hz, 2H).

MS: [M+H]⁺ 972.3

Synthesis of JM 360

A suspension of Ir-dimer (150 mg, 0,065 mmol), picolinic acid (17 mg,0.137 mmol) and Na₂CO₃ (70 mg, 0.65 mmol) in 20 mLdichloromethane/ethanol (4:1) was refluxed overnight. After coolingdown, the mixture was concentrated to dryness. The residue was purifiedby flash cromatography in dichloromethane/MeOH (gradient from 100:0 to10:1). The compound was recrystallized in dichloromethane/Et₂O. Yield:30%.

NMR: ¹H NMR (300 MHz, CDCl₃) δ 9.06 (d, J=8.2 Hz, 1H), 8.98 (d, J=8.1Hz, 1H), 8.61 (m, 3H), 8.46-8.21 (m, 4H), 8.13 (d, J=8.9 Hz, 1H), 7.83(m, 4H), 7.61 (m, 2H), 7.57-7.41 (m, 3H), 7.30 (d, J=7.2 Hz, 1H),7.24-7.12 (m, 1H), 6.89 (t, J=7.2 Hz, 1H), 6.76 (dd, J=8.9, 2.5 Hz, 1H),6.61 (dd, J=8.8, 2.6 Hz, 1H), 6.54 (d, J=2.5 Hz, 1H), 5.99 (d, J=2.6 Hz,1H), 3.85-3.41 (m, 32H), 3.34 (s, 3H), 3.33 (s, 3H).

MS: [2M+2Na]²⁺ calc. 1258.4012. found 1258.4030. [M+H]⁺ calc. 1236.4197.found 1236.4227.

Example 5 ECL with a Novel Iridium Complex

The electrochemiluminescence signal of several metal complexes wasassessed in an ELECSYS® analyzer (Roche Diagnostics GmbH). Measurementswere carried out homogeneously in the absence of streptavidin-coatedparamagnetic microparticles. Stock solutions of each metal complex at0.1 mg/ml DMSO were diluted with PBS buffer resulting in 10 nMsolutions. The 10 nM solutions were handled as samples on the ELECSYS®analyzer. 20 μl sample was incubated together with 90 μl Reagent 1(ProCell) and 90 μl Reagent 2 (ProCell) for 9 min at 37° C. andsubsequently the electrochemiluminescence signal was quantified.

ECL Results:

Reference Ru(bpy)3=10000 counts in 10 nmolar concentration

JM 360=31258 counts 10 nmolar concentration

RC 72=45512 count in 10 nmolar concentration

Example 6 Synthesis of an Iridium Complex with Reactive Group forBioconjugation

Ir(6-phenylphenanthridine)₂ 2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid (15 mg) was dissolved in a mixture of dry acetonitrile 5 mL and drypyridine 0.01 mL. Disuccinimidyl carbonate (DSC) (1.5 eq) was added andthe mixture was stirred under nitrogen at room temperature overnight.The solution was added to chloroform (10 mL), washed with 0.5 M HCl (1×2mL), saturated aqueous NaHCO₃ (1×2 mL) and water (2×5 mL) dried overMgSO₄, and concentrated in vacuo to yield a red powder.

Ir(6-phenylphenanthridine)₂ 2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid N-succinimidyl ester

¹H-NMR (400 MHz, CD₃CN) δ 9.25 (m, 1H), 9.17 (d, J=8.0 Hz, 1H), 8.83 (d,J=8.4 Hz, 1H), 8.75-8.68 (m, 1H), 8.60-8.54 (m, 3H), 8.47 (d, J=8.1 Hz,1H), 8.43 (d, J=2.1 Hz, 1H), 8.30 (d, J=8.1 Hz, 1H), 8.06 (t, J=15.4,7.2 Hz, 1H), 7.97-7.95 (m, 3H), 7.77-7.70 (m, 2H), 7.61 (t, J=15.2, 7.0Hz, 1H), 7.52-7.44 (m, 3H), 7.36 (d, J=8.3 Hz, 2H), 7.18 (t, J=15.2, 7.0Hz, 1H), 7.12-7.09 (m, 3H), 7.04-6.98 (m, 2H), 6.78 (t, J=14.9, 7.2 Hz,1H), 6.71 (t, J=14.8, 7.5 Hz, 1H), 6.57 (d, J=7.6 Hz, 1H), 3.07-3.01 (m,4H), 2.80 (s, 4H).

MS: [M+H]⁺ 1069.3

Example 7 Synthesis of an Iridium-Complex Conjugate with Biotin

Ir(6-phenylphenanthridine)₂ 2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid NHS ester (12 mg) and 4 mg ofN-Biotinyl-3,6-dioxaoctane-1,8-diamine trifluoroacetate was dissolved ina dry DMF 5 mL. Pyridine (0.016 mL in 2 mL DMF) was added and themixture was stirred under nitrogen at room temperature overnight. Thesolution was added to chloroform (10 mL), washed with 0.5 M HCl (1×2mL), saturated aqueous NaHCO₃ (1×2 mL) and water (2×5 mL) dried overMgSO₄, and concentrated in vacuo to yield a red powder. The product waspurified by column chromatography (silica, n-hexane/ethyl acetate) togive red powder.

MS: [M+H]⁺ 1328.6

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

While this disclosure has been described as having an exemplary design,the present disclosure may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the disclosure using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within the known orcustomary practice in the art to which this disclosure pertains.

What is claimed is:
 1. An iridium-based chemiluminescent compound ofFormula I

wherein R1-R16 is hydrogen, halide, cyano- or nitro-group, amino,alkylamino, substituted alkylamino, arylamino, substituted arylamino,alkylammonium, substituted alkylammonium, carboxy, carboxylic acidester, carbamoyl, hydroxy, substituated or unsubstituated alkyloxy,substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or R17, wherein R17 is aryl,substituted aryl, alkyl, substituted alkyl branched alkyl, substitutedbranched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substitutedalkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,wherein the substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstituatedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate or, wherein within R1-R12 and/orwithin R13-R16, respectively, two adjacent Rs can form an aromatic ringor a substituted aromatic ring, wherein the substituent is selected fromhydrogen, halide, cyano- or nitro-group, a hydrophilic group, likeamino, alkylamino, substituted alkylamino, alkylammonium, substitutedalkylammonium, carboxy, carboxylic acid ester, carbamoyl, hydroxy,substituted or unsubstituted alkyloxy, substituted or unsubstitutedaryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfino, sulfeno,sulfonamide, sulfoxide, sulfodioxide, phosphonate, phosphinate or,wherein within R1-R12 or/and within R13-R16, respectively, two adjacentRs can form an aliphatic ring or a substituted aliphatic ring, whereinthe substituent is selected from hydrogen, halide, cyano- ornitro-group, a hydrophilic group, like amino, alkylamino, substitutedalkylamino, alkylammonium, substituted alkylammonium, carboxy,carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstitutedalkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl,arylsulfonyl, sulfo, sulfino, sulfeno, sulfonamide, sulfoxide,sulfodioxide, phosphonate, phosphinate and wherein at least one ofR13-R16 is -Q-Y, wherein Q represents a linker and Y is a functionalgroup and wherein the linker Q is a straight or branched saturated,unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a 1 to20 atom chain with a backbone consisting of carbon atoms and one or moreheteroatoms selected from O, N and S.
 2. The compound according to claim1, wherein the linker Q is a saturated C1-C12 alkyl chain or a 1 to 12atom chain with a backbone consisting of carbon atoms and one or moreheteroatoms selected from O, N and S.
 3. The compound according to claim1, wherein the functional group Y is selected from the group consistingof carboxylic acid, N-hydroxysuccinimide ester, amino group, halogen,sulfhydryl, maleimido, alkynyl, azide and phosphoramidite.
 4. Aconjugate comprising a compound according to claim 1 and covalentlybound thereto an affinity binding agent.
 5. The conjugate of claim 4,wherein the affinity binding agent is selected from the group consistingof antigen and antibody, biotin or biotin analogue and avidin orstreptavidin, sugar and lectin, nucleic acid or nucleic acid analogueand complementary nucleic acid and receptor and ligand.
 6. The conjugateaccording to claim 4, wherein said affinity binding agent is a nucleicacid or an antibody.
 7. A method for measuring an analyte by an in vitromethod, the method comprising the steps of: a) providing a samplesuspected or known to comprise the analyte; b) contacting said samplewith a conjugate according to claim 4 under conditions appropriate forformation of an analyte conjugate complex; and c) measuring the complexformed in step (b) and thereby obtaining a measure of the analyte. 8.The method of claim 7, wherein said step of contacting is performed inan aqueous solution.
 9. The method of claim 7, wherein the affinitybinding agent is selected from the group consisting of antigen andantibody, biotin or biotin analogue and avidin or streptavidin, sugarand lectin, nucleic acid or nucleic acid analogue and complementarynucleic acid and receptor and ligand.
 10. The method of claim 7, whereinthe affinity binding agent of the conjugate is a nucleic acid or anantibody.